The sequencing of nucleic acids is one the most powerful and valuable tools for scientific research. As evidenced by the Human Genome project, there is an ever increasing demand for nucleic acid sequence information. There are numerous methods available for sequencing of nucleic acids. The first methods were developed almost twenty years ago. For example, the Sanger enzymatic (i.e., dideoxy chain termination) method involves synthesis of a DNA strand from a single-stranded template by a DNA polymerase. The Maxam and Gilbert method involves chemical degradation (i.e. chemical cleavage) of the original DNA. Both methods produce populations of radio-labelled polynucleotides that begin at a fixed point in the DNA to be sequenced and terminate at points which are dependent upon the location of a particular base in the original DNA strand. These polynucleotides are separated by a polyacrylamide gel electrophoresis, and the order of the nucleotides in the original DNA is directly read from an autoradiograph of the gel. However, the time-consuming electrophoresis step associated with these methods is difficult to perform in a highly parallel (i.e. greater than 1000 samples at a time per instrument) fashion.
Although both the Sanger and Maxam-Gilbert methods are currently used, there have been many changes and improvements. The enzymatic chain termination method is probably the most popular and widely used technique for sequence determination, especially since the automation of the procedure has been accomplished through use of fluorescent, rather than radioactive labelling, and the utilization of amplification technology. The incorporation of amplification technology (e.g., the polymerase chain reaction [PCR]) enables the sequencing reaction to be cycled. Other advances include sequencing by chemiluminescence, multiplexing, and solid phase sequencing.
Other nucleic acid sequencing methods, such as sequencing by hybridization and pyrosequencing, have been developed that eliminate the electrophoresis step associated with the Sanger and Maxam and Gilbert methods, thereby allowing more samples to be sequenced in parallel. However, such methods often involve either lengthy cloning and amplification steps, or a time-consuming chemical cleavage step wherein a fluorescently-labeled polynucleotide is removed by enzymatic digestion.
Therefore, what is need is are compositions and methods that reduce the complexity of and time-consuming nature of parallel nucleic acid sequencing.